The events regulating the earliest stages of hematopoietic commitment and development in humans are poorly understood, given the inaccessibility of the early embryo. The capacity of embryonic stem (ES) cells to differentiate in culture and recapitulate the critical programs responsible for early blood cell development provides a powerful model for gaining a better understanding of these processes. Studies with mouse ES cells have demonstrated that regulation of hematopoietic development in ES cell-derived embryoid bodies (EBs) is similar to that of the early embryo. The experiments in this proposal will translate findings from the mouse system to human ES cell differentiation with the ultimate goal of establishing a model for investigating the earliest stages of human hematopoietic development. In the first aim of this proposal, different stages of hematopoietic development will be characterized within the developing human EBs. These analyses are essential, as they will define the developmental program that gives rise to the human hematopoietic system. The focus of the second aim is to define the molecules that regulate blood cell development in human EBs. Uncovering the key factors in this process is important, as these findings will provide the first insights into the regulation of early human hematopoietic development. In addition, this information will enable us to establish optimal protocols for hematopoietic development within the EBs. The goal of the third aim of the proposal is to identify and characterize the long-term repopulating stem cell (LTRSC) in the human EBs. While challenging, these studies are important, as the identification of LTRSC in these cultures would provide a model system for investigating events that regulate their generation, self-renewal and maturation. In addition, the development of LTRSC from EBs would provide a novel source of these cells for transplantation. The studies in the first three aims will be carried out with the two different cell lines, WA01 and ES02. In the final aim, we will compare the hematopoietic potential of 12 different huES cell lines, to determine the efficiency of each to generate the populations identified in the previous three aims. The following lines will be used for this aim; WA01, WA07, WA09, ES02, ES03, ES04, TE-03, TE-06, MI01, UC01, BG02, BG03. Findings from these studies will provide new and important information on the origins and developmental regulation of the human hematopoietic system and in doing so will identify the hemangioblast, the primitive erythroid lineage and the earliest stages of definitive hematopoiesis. This information will provide the first insights into the regulation of mesoderm induction and hematopoietic specification during human embryonic development. Generation of LTRSC from human ES cells could ultimately expand the number of patients and the spectrum of diseases treated by hematopoietic stem cell transplantation.